Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of nateglinide, cilostazol and its active metabolite 3,4-dehydro-cilostazol in Wistar rat plasma and its application to pharmacokinetic

Nateglinide (NTG), an insulin secretogogue, has been studied in rats for drug–drug interaction with cilostazol (CLZ), an antiplatelet agent commonly used in diabetics. We developed a liquid chromatography tandem mass spectrometry (LC–MS/MS) based method that is capable of simultaneous monitoring plasma levels of nateglinide, cilostazol, and its active metabolite 3,4-dehydro-cilostazol (DCLZ). All analytes including the internal standard (Repaglinide) were chromatographed on reverse phase C18 column (50 mm × 4.6 mm i.d., 5 μm) using acetonitrile: 2 mM ammonium acetate buffer, pH 3.4 (90:10, v/v) as mobile phase at a flow rate 0.4 ml/min in an isocratic mode. The detection of analyte was performed on LC–MS/MS system in the multiple reaction monitoring (MRM) mode. The quantitations for analytes were based on relative concentration. The method was validated over the concentration range of 20–2000 ng/ml and the lower limit of quantitation was 20 ng/ml. The recoveries from spiked control samples were >79% for all analytes and internal standard. Intra- and inter-day accuracy and precision of validated method were with in the acceptable limits of <15% at all concentration. The quantitation method was successfully applied for simultaneous estimation of NTG, CLZ and DCLZ in a pharmacokinetic drug–drug interaction study in Wistar rats.